JP2618727B2 - Method for quantifying test components in whole blood - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] The present invention relates to the activity of a test component in whole blood using a dry analytical element for the quantification of a test component in a biological fluid such as plasma or serum. It relates to a method for quantitatively analyzing a value.

[Prior Art] Measuring the concentration or activity of a test component (analyte) in a body fluid, particularly in blood, is important in clinical medicine, and is used, for example, for diagnosing a disease and tracking the course of treatment. Have been. Recently, the use of dry chemical analysis elements has been increasing because of their simplicity, speed and economical use. The dry chemical analysis is a chemical analysis using a test piece or a multi-layer analysis element in which the analysis reagent is introduced in a dry state, unlike the conventional chemical analysis by a wet method using a solution-type analysis reagent. Dry analysis elements are, for example, JP-B-53-21677, JP-A-55-164356,
It is known from JP-A-60-222769. As an example, the dry multi-layer analysis element has a configuration having a transparent support, a reagent layer, and a developing layer. The transparent support is, for example, a light-permeable, water-impermeable, thin organic polymer sheet. The reagent layer applied on the transparent support reacts with the test component in the body fluid sample,
An analysis reagent that shows a change in signal according to the amount of the test component, for example, a change in optical density due to coloring of a dye is included.
The spreading layer uniformly spreads the body fluid sample so as to have an area approximately proportional to the amount of the spotted body fluid sample.

In order to perform quantitative analysis using a dry multi-layer analysis element, an appropriate amount of a body fluid sample is spotted (dropped or adhered) on a display of a development layer. The body fluid sample uniformly developed in the developing layer reaches the reagent layer, where the analysis reagent reacts with the test component in the body fluid sample to change the signal. During the course of the reaction, the analytical element is kept at a constant temperature (incubation). The change in signal that occurs after the reaction has proceeded sufficiently for an appropriate amount of time is read.
That is, when the signal is coloring by a dye, the reagent layer is irradiated with irradiation light from the transparent support side, the amount of reflected light is measured at a specific wavelength, and the optical density of the coloring is read. The change in the read signal is calculated in advance based on a calibration curve indicating the relationship between the amount of change in the signal and the amount of the test component in the body fluid sample. Converted (colorimetric method). The change in the signal to be read may be the absolute value of the signal after a certain time after the liquid sample is spotted, or the amount of change per unit time in the certain time.

When the body fluid sample is blood, many dry analysis elements that have been proposed or are conventionally used cannot directly use whole blood as a sample, and separate and remove red blood cells from blood before analysis to remove serum. Alternatively, an operation for obtaining plasma (generally, a centrifugation operation) was required. Despite the simplicity, speed, and economy of analysis using dry analytical elements, it requires a large amount of labor, time, and equipment cost to separate and remove red blood cells before performing the analysis operation. This has reduced the usefulness of dry analytical elements by half.

In order to perform quantitative analysis using whole blood as a sample using a dry analytical element, it is necessary to separate and remove blood components typified by red blood cells from the blood sample by any means or reaction in the analytical element to obtain serum or plasma. At the same time, it is required that the red dye of the separated red blood cells does not interfere with the measurement of the optical density. Japanese Patent Publication No. 53-21677 discloses that a filtration layer is provided in an analysis element in order to separate and remove blood cell components in blood. However, it takes a very long time to obtain serum or plasma by separating and removing blood cell components from whole blood using a filtration layer provided in the analysis element. Further, a part of the target test component may be lost during the filtration, or red blood cells may be lysed during the filtration, resulting in inaccurate analysis.

A dry analytical element useful for quantitative analysis of test components in whole blood samples, which rapidly separates and removes red blood cells in blood and rapidly diffuses test components in plasma obtained to the reagent layer. And JP-A-62-138756. However, with this analysis element, it turned out that even if the blood sample had the same concentration of the target test component in plasma, the analysis results differed depending on the hematocrit value of the blood sample, and accurate analysis could not be performed. did. On the other hand, Japanese Patent Application Laid-Open No. 64-54354 proposes that a white colorant be contained in order to block the irradiation light for the measurement and avoid the influence of the red pigment of the red blood cells. It is considered that providing a light-shielding layer for avoiding the effect of the red blood cell dye in the analysis element is effective for performing accurate analysis. However, in order to adjust the shading with the colorant, it is necessary to control the particle size and the dispersion state of the colorant at the time of production, which complicates the production process and increases costs. It has also been found that the blood cell component cannot be sufficiently separated by providing the light-shielding layer, or, on the contrary, the separated plasma component cannot be sufficiently supplied to the reagent layer.

[Problems to be Solved by the Invention] A technical problem of the present invention is that a blood cell component in a whole blood sample is rapidly separated and removed by using a dry-type multi-layered analysis element, and the separated blood cell component is colored. It is to provide a method for quantitative analysis of a test component contained in a whole blood sample in which the analysis is not hindered by a dye, whereby the quantitative analysis of the test component in the whole blood sample can be performed without depending on the hematocrit value. To be able to do it quickly and accurately.

[Means for Solving the Problems] The technical problem of the present invention is to provide an optical density which can be detected on a water-impermeable light-permeable support at least as a result of a reaction with a test component in whole blood. A colorimetric measurement method using a multi-layer analysis element in which a coloring reagent layer giving a change and a blood cell separation layer for separating and removing blood cell components from whole blood and supplying plasma to the reagent layer are stacked in this order. In the method of quantifying a test component, a whole blood sample is spotted on the uppermost layer of a blood cell separation layer, and the colored dye of the blood cell component separated from the whole blood spotted by the blood cell separation layer, on the support side After the reflected optical density value detected from becomes substantially constant, the amount of change in the reflected optical density per unit time in the reagent layer is a wavelength in a range overlapping with the reflected light indicated by the colored dye of the blood cell component. Measured from the support side with The density value or activity value of the test component of whole blood from the variation of the reflection optical density per has been solved by obtaining a colorimetric assay.

[Detailed Description of Means for Solving the Problems] As a multi-layer analysis element used in the quantitative analysis method of the present invention, separation and removal of blood cell components from whole blood is performed by reacting with test components in whole blood. The optical density change in the reagent layer as a result of the completion is completed before the detection is started, the reflected optical density detected from the transparent support side of the colored dye of the separated blood cell component becomes constant, and the separated blood component It is preferable that the colored dye can be sufficiently detected optically from the transparent support side. Further, in the multi-layer analysis element, it is preferable that the reagent layer and the blood cell separation layer are bonded and integrated by a partially disposed adhesive so as not to prevent uniform passage of the liquid.

The dry-type multi-layer analysis element used in the method of the present invention is basically a portion showing a signal change according to the amount of the test component due to the reaction with the test component in the whole blood sample (the base of the analysis component). ) And a part (blood cell separation layer) that separates and removes blood cell components from a whole blood sample and supplies plasma to the base of the analysis element.

Among them, as the base of the analysis element, a dry integrated multi-layer analysis element for serum / plasma samples which has been known can be used. That is, the color reagent layer and the developing layer are laminated and integrated in this order on the transparent support. A detection layer or a water absorbing layer may be provided between the support and the color reagent layer. If the shape and function of the analytical element can be retained in layers other than the support of the multilayer analytical element, the support is not essential, or the support is present when measuring the optical density of color inside the element. It may be configured so that it can be separated from the color reagent layer (or the detection layer or the water absorbing layer).

Preferably, the transparent support is water impermeable. Preferred materials include polyethylene terephthalate, polycarbonate of bisphenol A, and cellulose esters (eg, cellulose triacetate, cellulose acetate butyrate). These supports are provided with hydrophilicity on the surface and are subjected to a physicochemical activation treatment (eg, a physicochemical activation treatment) in order to strengthen adhesion to a layer containing a hydrophilic polymer binder such as a coloring reagent layer, a water absorbing layer, and a detection layer. , Corona discharge treatment) and hydrophilic treatment such as providing an undercoat layer.

The reagent composition includes a substance (a color reagent composition) capable of producing a substance (eg, a coloring reagent composition) capable of forming a dye (dye) optically detectable in the presence of a test component. Reagent composition for producing a dye by oxidation of the dye (eg, triarylimidazole leuco dye described in U.S. Pat. No. 4,089,747, diarylimidazole leuco dye described in JP-A-59-193352, etc.), diazonium salt, oxidation A composition containing a chromogenic compound that forms a dye upon coupling with a coupler (eg, a combination of 4-aminoantipyrines and phenols or naphthols),
A composition comprising a compound capable of producing a dye in the presence of a reduced coenzyme and an electron transfer agent, or the like can be used. In the case of an analytical element for measuring an enzyme activity, for example, a composition containing a self-developing substrate capable of releasing a colored substance such as p-nitrophenol or p-nitrophenyl phosphate can also be used. The contents of these reagent compositions are selected according to the test components in the whole blood sample.

The reagent composition may contain all the components in one layer, or may contain the components in a plurality of layers. For example, a hydrophilic polymer solution containing a part or the whole of the reagent composition is applied and dried on the surface of the support subjected to the hydrophilic treatment, and the hydrophilic polymer containing a part or the whole of the reagent composition is bound. Agent
There is a method of forming a substantially uniform layer. Examples of the hydrophilic polymer include gelatin and derivatives thereof (eg, phthalated gelatin), cellulose derivatives (eg, hydroxypropylcellulose), agarose, acrylamide polymers, methacrylamide polymers, acrylamide or methacrylamide, and various vinyl monomers. And the like. A porous layer not containing a reagent composition is adhered on a uniform layer containing a part or all of a reagent composition using a hydrophilic polymer as a binder by a method as described in JP-A-55-164356. After that, a solution or dispersion containing a part or all of the reagent composition may be applied in a porous manner to contain the reagent composition.

The bonding and lamination of the porous layer may be performed via the bonding layer. The adhesive layer is preferably made of a hydrophilic polymer capable of adhering the porous layer when swollen with water, for example, gelatin, gelatin derivatives, polyacrylamide and the like. Further, a part of the reagent composition can be included in the adhesive layer.

Since the porous layer also functions as a developing layer under the analytical element, it is preferably a layer having a liquid measuring action. The liquid metering action means that the supplied liquid sample is applied to the surface of the spreading layer in a dot-like manner, and the components contained therein are not substantially ubiquitous, and the surface is almost uniformly occupied per unit area in the direction of the surface. This is the action of spreading the film uniformly at a constant rate. In the multilayer analytical element used in the method of the present invention, it means an action of spreading the plasma component passing through the blood cell separation layer at a substantially constant rate per unit area in the surface direction.

Either a non-fibrous porous layer or a fibrous porous layer can be applied as the porous layer. As the non-fiber porous layer, a brush polymer composed of cellulose esters (eg, cellulose acetate, cellulose acetate / butyrate, cellulose nitrate) described in JP-B-53-21677, US Pat. No. 3,992,158, US Pat. No. 1,421,341, etc. Are preferred. Also, microporous membranes such as polyamides such as 6-nylon and 6,6-nylon, polyethylene, polypropylene and the like, and microporous membranes composed of polysulfone described in JP-A-62-27006 can be used.

As a material constituting the fibrous porous layer, filter paper, nonwoven fabric, woven fabric (eg, plain woven fabric), knitted fabric (eg, tricot knitted fabric), glass fiber filter paper, and the like can be used. Of these, woven and knitted fabrics are preferred. The woven fabric or the like may be subjected to a physicochemical treatment (eg, glow discharge treatment) described in JP-A-57-66359.

In the porous layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770, JP-A-63-219397, JP-A-63-19397
It may contain a hydrophilic polymer or a surfactant as described in JP 112999 and JP-A-62-182562.

Conventional multi-layer analysis elements include light-reflecting / light-blocking fine particles such as titanium dioxide and barium sulfate dispersed in a porous layer, or hydrophilic such as gelatin containing dispersed light-reflecting / light-blocking fine particles. Placing a conductive polymer binder layer is performed, but is not necessary as a base for the analytical element of the present invention. The underlayer of the multi-layer analysis element can be used as an analysis element for serum / plasma. By selecting a reagent composition to be contained, for example, AST (aspartate aminotransferase), ALT (alanine aminotransferase), LDH (lactate dehydrogenase),
It can provide a base for analytical elements for creatine kinase and amylase.

As the blood cell separation layer, a fibrous porous layer and a non-fibrous porous layer that are bonded and integrated by a partial bonding method using a halftone printing method (eg, a gravure printing method, a screen printing method) are effective. . A typical example of the partial bonding method using a gravure printing method is to apply a hot-melt adhesive in a molten state by heating to a high temperature on one surface of a fibrous porous layer or a non-fibrous porous layer. Immediately after they are attached in a dot form by transfer from a gravure roller, they are overlapped and passed through a laminating roller to bond and integrate. As this method, the method described in JP-A-62-138756 is effective. In the blood cell separation layer thus obtained, the fibrous porous layer and the non-fibrous porous layer are bonded and integrated by an adhesive partially disposed so as not to hinder the passage of liquid. The blood cell separation layer is arranged so that the fibrous porous layer is on the upper side (the side on which the whole blood sample is supplied by spotting).

Since the fibrous porous layer of the blood cell separation layer also functions as a developing layer when a whole blood sample is spotted on the surface thereof, it is preferably a layer having a liquid measuring action.

As a material constituting the fibrous porous layer, filter paper, nonwoven fabric, woven fabric (eg, plain woven fabric), knitted fabric (eg, tricot knitted fabric), glass fiber filter paper, and the like can be used. Of these, woven and knitted fabrics are preferred. The woven fabric or the like may be subjected to a physicochemical treatment (eg, glow discharge treatment) described in JP-A-57-66359.

The fibrous porous layer may contain a hydrophilic polymer, a surfactant or the like in order to adjust the development area and the development speed. However, in this case, the hydrophilic polymer surfactant used is selected from those which do not cause erythrocyte hemolysis in the layer. In addition, the fibrous porous layer may contain a hydrophilic polymer or an inorganic salt that promotes blood cell separation.

As the non-fiber porous layer, a brush polymer composed of cellulose esters (eg, cellulose acetate, cellulose acetate / butyrate, cellulose nitrate) described in JP-B-53-21677, US Pat. No. 3,992,158, US Pat. No. 1,421,341, etc. Are preferred. Also, 6-nylon,
Microporous membranes such as polyamides such as 6,6-nylon, polyethylene and polypropylene, and microporous membranes composed of polysulfone described in JP-A-62-27006 can also be used.

When the non-fibrous porous layer is a membrane filter made of a so-called brush polymer made by a phase separation method, the liquid passage in the thickness direction is most likely to be on the free surface side (glossy surface) in the production of the membrane. Normally, it is narrow, and the hole diameter when the cross section of the liquid passage path is approximated to a circle is
It is smallest near the free surface. This type of brush polymer is preferred for non-fibrous porous layers used in blood cell separation layers. When a brush polymer membrane is used for the blood cell separation layer, the membrane is adhered and integrated such that the glossy surface of the membrane faces away from the fibrous porous layer of the blood cell separation layer. With this configuration, the liquid passage in the thickness direction of the non-fibrous porous layer becomes narrower as the distance from the fibrous porous layer of the blood cell separation layer increases.

The effective pore size of the non-fibrous porous layer used in the blood cell separation layer is preferably in the range of about 0.80 μm to about 30 μm. The effective pore size referred to in the present specification is indicated by the pore size measured by the limiting bubble pressure method (valve point method) according to ASTM F-316-70. The effective pore size of the non-fibrous porous layer of the blood cell separation layer is too large. The separation and removal of blood cell components from the whole blood sample were not performed completely, and some of the blood cell components reached the base of the multi-layer analysis element, and were not included in the test components in the whole blood sample and the base of the multi-layer analysis element. The reaction with the used reagent composition is hindered and accurate quantitative analysis cannot be performed. On the other hand, if the effective pore size is too small,
When a large share (shear strain) is applied to red blood cells while moving through the non-fibrous porous layer, hemolysis occurs, the components in the red blood cells are released, and the concentration and activity value of the test component in the whole blood sample changes However, accurate quantitative analysis cannot be performed.

In the non-fibrous porous layer used for the blood cell separation layer, a difference in the moving speed between a blood cell component represented by red blood cells and a plasma component occurs while a whole blood sample moves therein. In addition, it is necessary to select an effective pore diameter such that resistance is applied to blood cell components to such an extent that hemolysis of red blood cells does not occur.

In order to laminate and integrate the base of the above-described analysis element and the blood cell separation layer, the above-described bonding and integration method by a partial bonding method using halftone printing (eg, gravure printing, screen printing) is preferable. When the hot-melt adhesive is used for the gravure printing method, the hot-melt adhesive in a molten state by heating to a high temperature may be transferred to either the base of the multilayer analysis element or the blood cell separation layer. The partial adhesion is performed between the underlying porous layer of the multilayer analysis element and the non-fibrous porous layer of the blood cell separation layer.

The multilayer analytical element used in the analysis method of the present invention has an optical density change detectable as a result of a reaction with a test component in a whole blood sample on an impermeable light-transmitting support (transparent support). A blood cell separation layer with the function of separating and removing blood cell components from whole blood and supplying plasma to the color reagent layer on the base of the analytical element provided with the color reagent layer to be applied is laminated and integrated. It is. However, this analytical element does not have a function such as a so-called light-shielding layer or light-reflecting layer for preventing the colored dye of the blood cell component to be separated and removed from being substantially detected from the transparent support side. Is the feature.

Quantitative analysis of a test component in a whole blood sample using the multilayer analysis element having the above-described configuration is performed as follows.

A certain amount (eg, 20 μL) of a whole blood sample collected from a human is spotted on the surface of the fibrous porous layer which is the uppermost layer of the blood cell separation layer which is the surface farthest from the transparent support of the analysis element. The spotted whole blood sample is spread almost uniformly in the surface direction in the fibrous porous layer. At the same time, the whole blood sample moves downward, where the fibrous porous layer of the blood cell separation layer and the fibrous porous layer work together to convert the whole blood sample into blood cell components and plasma. Separated, the blood cell components remain completely retained up to the lower surface of the non-fibrous porous layer of the blood cell separation layer, and substantially only the plasma migrates to the underlying porous layer of the underlying analytical element. To go. When the separated plasma is supplied to the porous layer underlying the analysis element, the plasma is spread so as to be substantially constant in both directions according to the amount thereof.

In the above-described multilayer analytical element, the blood cell component in the whole blood sample is substantially completely separated up to the lower surface of the non-fibrous porous layer of the blood cell separation layer, and the porous layer underlying the analytical element is substantially separated. It is strange that it never reaches, and the reason has not been clarified yet. It is clear, however, that the mechanism is not so-called physical filtration, which separates blood cell components by their size. Attempts to physically filter blood cell components from whole blood usually involve a large share of red blood cells, causing hemolysis. However, in the above-mentioned multilayer analytical element, hemolysis of erythrocytes is not observed, as will be shown in the later examples. The separation and removal of the blood cell component in the blood cell separation layer is completed immediately after the whole blood sample is spotted on the fibrous porous layer of the blood cell separation layer. Separation and removal of blood cell components at the latest after application of whole blood sample
Completed within 30 seconds. The multi-layer analysis element includes:
Since it does not have a light-shielding function to prevent the colored dye of the separated blood cell component from being detected from the transparent support side, the colored dye of the blood cell component (red dye of red blood cells) ) Starts to be optically detected from the transparent support side. However, the reflection optical density of the colored dye of the blood cell component detected from the transparent support is constant at an absolute value determined by the hematocrit value of the whole blood sample and other individual differences at the same time as the separation of the blood cell component is completed. And found that it did not change thereafter. This is a surprising phenomenon.

After separation and removal of the blood cell component, the test component in the plasma, which has been spread almost uniformly in the porous layer underlying the analysis element, reacts with the color reagent composition contained in the analysis element base. Shows a change in the optically detectable signal, independent of the optical density of the colored dye of the separated blood cell component.

To perform a quantitative analysis of the test component in the whole blood sample using the multilayer analysis element, after the reflection optical density of the colored dye of the separated blood cell component detected from the transparent support side becomes constant, The reflection optical density of the color produced as a result of the reaction between the test component and the reagent composition in the whole blood sample is measured. Using the measured optical density as a parameter, the spotted whole blood sample is determined based on the relationship between the amount of the test component and the parameter (normally, a calibration curve) which has been measured in the same manner in advance. The amount of the test component is detected. Used as a parameter for colorimetry.

At this time, the parameter used is a change value of the reflection optical density per unit time during a certain time after spotting, and it is not necessary to add any correction to the measured value.

If the multilayer analytical element does not have a transparent support, or if the support is to be peeled off, instead of reflection measurement of the color in the element through the transparent support, a color reagent layer,
Reflection photometry may be performed from a layer opposite to the blood cell separation layer on which the whole blood sample is spotted, such as a water absorption layer and a detection layer.

The amount of the whole blood sample required for the analysis method of the present invention is, for example,
About 5 μL to about 30 μL, preferably about 8 μL to about 15 μL
L. In the analysis method of the present invention, the reflection optical density value of the colored dye of the blood cell component separated from the spotted whole blood by the blood cell separation layer, which is detected from the support side, is substantially constant (at most at most). After about 30 seconds, usually within about 20 seconds), within a range of about 20 ° C. to about 40 ° C., preferably 37 ° C.
While incubating at a substantially constant temperature in the vicinity of ° C., the amount of change in the reflection optical density per unit time in the reagent layer is measured from the support side for a time range of about 1 minute to about 10 minutes. The concentration value or activity value of the test component is determined according to the principle described above. The analysis method of the present invention is described in JP-A-60-125543,
A high-precision quantitative analysis can be performed by a simple operation using the chemical analyzer described in JP-A-60-220862, JP-A-61-294367, JP-A-58-161867, and the like.

[Effects of the Invention] According to the quantitative analysis method of the present invention, the quantitative analysis of the test component in the whole blood sample does not depend on the hematocrit value, using a multilayer analysis element having no light shielding layer or light reflection layer, It can be performed accurately in a short time of several minutes (about 2 minutes to about 10 minutes). Further, the present invention is applied to the quantitative analysis of various test components in a whole blood sample simply by changing the reagent composition contained in the base of the multilayer analysis element used in the quantitative analysis method of the present invention to a reagent component corresponding to the test component. be able to.

 A preferred embodiment of the present invention is as follows.

(1) Separating blood cell components from whole blood on a water-impermeable, light-permeable support and at least a color reagent layer that gives a detectable optical density change as a result of a reaction with a test component in whole blood A method for quantifying a test component by a colorimetric method using a multi-layer analysis element laminated in this order, wherein the blood cell separation layer for removing and supplying plasma to the reagent layer is provided. The whole blood sample is spotted on the upper layer, and the reflection optical density value of the colored dye of the blood cell component separated from the whole blood spotted by the blood cell separation layer, which is detected from the support side, is substantially constant. After that, the amount of change in the reflection optical density per unit time in the reagent layer is measured from the support side at a wavelength in a range overlapping with the reflected light indicated by the colored dye of the blood cell component, and the obtained reflection per unit time is obtained. Test component in whole blood from change in optical density A method for quantifying a test component in whole blood by determining the activity value of the compound by colorimetry.

(2) An analysis in which the analysis element completes separation and removal of blood cell components from whole blood before a change in optical density in a reagent layer is started to be detected as a result of a reaction with a test component in whole blood. The method according to (1), which is an element.

(3) The method according to (1), wherein the analysis element is an analysis element capable of optically detecting a colored dye of a separated blood cell component from the support side.

(4) The analysis element is an analysis element in which after the separation and removal of the blood cell component from the whole blood is completed, the optical density detected from the support side of the colored dye of the separated blood cell component becomes substantially constant ( The method according to 1).

(5) The analysis element in which the reagent layer and the blood cell separation layer of the analysis element are adhered and integrated by an adhesive partially disposed so as not to prevent uniform passage of the liquid (1). Measurement method described in 1.

[Example 1] Multilayer analytical element for quantifying AST activity value 1-1 Preparation of base for analytical element Color development of the following components on a 180 μm thick polyethylene terephthalate (PET) colorless transparent smooth sheet subbed with gelatin The reagent layer was applied to a thickness of 15 μm after drying, and dried to form a coloring reagent layer.

Coating amount of the coloring reagent layer (per 1 m ² ) Deionized gelatin 20 g p-nonylphenoxypolyglycidol (containing an average of 10 glycidol units) 1.5 g Peroxidase 15000 IU FAD (flavin adenine dinucleotide) 22 mg
TPP (thiamine pyrophosphate) (cocarboxylase) 93 mg pyruvate oxidase 13000 IU 2- (3,5-dimethoxy-4-hydroxyphenyl) -4
-Phenethyl-5- [4- (dimethylamino) phenyl] imidazole (leuco dye) 280 mg (Apply an aqueous solution adjusted to pH 7.5 with a dilute NaOH aqueous solution) Then, adhere the following component coating amount on the color reagent layer The layer was applied from an aqueous solution so that the thickness after drying became 3 μm, and dried to form an adhesive layer.

At a rate of about 30 g / m ² on the adhesive layer coverage ⁽² per 1m) Deionized gelatin 4.0 g p-nonylphenoxy polyglycidol (average 10 glycidol unit content) 430 mg L-sodium aspartate 250mg then the adhesive layer of the Water was supplied to wet the entire surface almost uniformly, and a broad woven fabric made of PET (thickness: about 150 μm, void volume: 9.8 μL / m ² ) was lightly laminated, adhered, and dried.

Next, an aqueous solution of the AST detection reagent composition having the following composition was applied almost uniformly to the cloth at a rate of 100 mL / m ² , dried and impregnated to complete the base of the multilayer analytical element for AST activity determination.

Composition of aqueous solution of AST detection reagent composition Trishydroxymethylaminomethane 3.7 g Potassium phosphate 4.4 g α-Ketoglutaric acid 4.0 g Hydroxypropylmethylcellulose (methoxy group 28 to
30%, containing 7 to 12% of hydroxypropoxy group; viscosity of 2% aqueous solution at 20 ° C. 50 cps) 8.7 g octylphenoxypolyethoxyethanol (containing an average of 10 oxyethylene units) 27 g magnesium chloride 2.3 g oxaloacetate decarboxylase 200,000 IU Ascorbate oxidase 180,000 IU 880 g of water (adjusted to pH 7.5 with diluted NaOH aqueous solution) 1-2 Preparation of blood cell component separation layer Tricot knitted fabric (thickness about 250 μm) in which PET spun yarn equivalent to 50 denier is 36 gauge knitted Was impregnated with an aqueous solution having the following composition and dried.

Polyethylene glycol (average molecular weight: 50,000) 2.0 g Sodium tetraborate 2.0 g Water 96 g Next, the impregnated tricot knitted fabric is heated to a temperature of 80 ° C., and a hot-melt adhesive melted by heating to a temperature of 130 ° C. is applied to the surface. Then, the particles were attached in the form of dots by transfer from a gravure roller by a gravure printing method. The dot pattern of the gravure roller is a circle having a dot diameter of 0.3 mm, a center-to-center distance of dots of 0.6 mm, and a dot area ratio of about 20%.
The amount of adhesive adhered was about 2 g / m ² . Then, on the surface of the hot fabric immediately after the adhesive is transferred, the effective pore size 3.
A non-glossy surface of a cellulose acetate membrane filter having a thickness of 0 μm, a thickness of 140 μm, and a porosity of about 80% was passed through a lamination roller with the non-glossy surfaces facing each other. .

1-3 Completion of Multi-Layer Analysis Element This blood cell separation layer is adhered on the base of the analysis element prepared in the step 1-1 by the partial adhesion method by the gravure printing method similar to the step 1-2, and integrated. I let it.

In the same manner as in the step 1-2, the hot-melt type adhesive that was heated and melted was attached to the surface of the membrane filter of the blood cell separation layer in the form of dots by the gravure printing method, and then immediately prepared in the step 1-1. The analysis element was opposed to the ground side of the broad woven cloth, and both were passed through a laminating roller to be laminated and bonded and integrated.

The completed multilayer analysis element was cut into a square chip having a side of 15 mm and placed in an organic polymer slide frame described in JP-A-57-63452 to complete a multilayer analysis slide for quantitative analysis of AST activity.

Example 2 Multilayer Analytical Element for Quantifying ALT Activity Value 2-1 Preparation of Base for Analytical Element A color forming reagent layer having the following component coverage was dried on a 180 μm thick PET colorless transparent smooth sheet undercoated with gelatin. It was applied to a thickness of 15 μm and dried to form a color reagent layer.

Coating amount of coloring reagent layer (per 1 m ² ) Deionized gelatin 20 g p-nonylphenoxypolyglycidol (containing an average of 10 glycidol units) 1.5 g Peroxidase 15000 IU FAD (flavin adenine dinucleotide) 22 mg
TPP (thiamine pyrophosphate) (cocarboxylase) 93 mg birubic acid oxidase 13000 IU 2- (3,5-dimethoxy-4-hydroxyphenyl) -4
-Phenethyl-5- [4- (dimethylamino) phenyl] imidazole (leuco dye) 280 mg (Apply an aqueous solution adjusted to pH 6.5 with a dilute NaOH aqueous solution) Then, adhere the following component coating amount on the color reagent layer The layer was applied from an aqueous solution so that the thickness after drying became 3 μm, and dried to form an adhesive layer.

Amount of adhesive layer (per 1 m ² ) Deionized gelatin 4.0 g p-nonylphenoxypolyglycidol (containing an average of 10 glycidol units) 160 mg (an aqueous solution adjusted to pH 7.0 with dilute NaOH aqueous solution) at a rate of about 30 g / m ² by supplying the water substantially uniformly wet the entire surface, by applying a light pressure the PET broad woven fabric (thickness: about 150 [mu] m, void volume 9.8μL / m ²⁾ was laminated to the And allowed to dry.

Next, an aqueous solution of an ALT detection reagent composition having the following composition was applied almost uniformly to the cloth at a rate of 100 mL / m ² , dried and impregnated to complete a base for a multilayer analytical element for ALT activity determination.

Composition of Aqueous Solution of ALT Detection Reagent Composition Trishydroxymethylaminomethane 2.2 g Potassium phosphate 4.5 g α-Ketoglutaric acid 4.0 g L-alanine 27.5 g Hydroxypropyl methylcellulose (methoxy group 28-
30%, containing 7 to 12% of hydroxypropoxy group; viscosity of 2% aqueous solution at 20 ° C. 50 cps) 8.7 g octylphenoxypolyethoxyethanol (average content of 10 oxyethylene units) 27 g magnesium chloride 2.4 g water 880 g (dilute NaOH aqueous solution) 1-3 The multi-layer analysis element was completed. A blood cell separation layer was prepared in the same manner as in step 1-2 of Example 1, and this blood cell separation layer and ALT prepared in step 2-1 were prepared. The base of the analysis element for activity measurement was laminated and integrated by the partial bonding method in the same manner as in the step 1-3 of Example 1 to complete a multi-layer analysis element. A multi-layer analytical slide for quantification was completed.

The obtained analytical element for measuring AST activity (Example 1) and ALT
The analysis element for activity measurement (Example 2) is composed of a transparent support, a coloring reagent layer and a broad woven fabric layer which are laminated and integrated in this order, a membrane filter layer and a tricot knitted fabric layer which are laminated and integrated in this order. This is a configuration in which a blood cell separation layer having a structured configuration is integrally laminated in this order.

The membrane filter layer and the tricot knitted fabric layer, which are laminated and integrated by the partial adhesion method, cooperate with each other to separate and remove blood cell components from the whole blood sample spot-supplied on the tricot knitted fabric as a specimen. At this time, the tricot knitted fabric layer also acts as a developing layer, and spreads the supplied whole blood sample in the direction of the surface at a substantially constant rate per unit area. The broad woven fabric layer is a reagent composition for detecting ALT or AST that generates pyruvate depending on the amount of ALT (in Example 2) of AST (in Example 1) in plasma that has passed through the blood cell separation layer. Acts as a reaction layer. At this time, the broad woven fabric layer also acts as a spreading layer, and spreads the plasma that has passed through the blood cell separation layer in the direction of the surface at a substantially constant rate per unit area. The coloring reagent layer adds pyruvate generated in the broad woven fabric layer in accordance with the amount of AST in (Example 1) and the amount of ALT in (Example 2) in the plasma component passed through the blood cell separation layer. It acts as a layer that converts and accumulates into a dye via hydrogen. The dye is detected optically through the transparent support.

When a whole blood sample was spotted on the surface of the tricot knitted fabric layer of the multilayer analytical element of Example 1 and Example 2, the blood cell component typified by red blood cells in the whole blood was substantially completely up to the membrane filter layer. It was observed that it was separated off and did not reach the broad woven fabric layer. However, since the multi-layered analytical element does not have a light shielding layer for preventing the red dye of the separated blood cell component from being detected from the transparent support side where the light is emitted, the blood cell component separated in the upper layer from the transparent support side is not provided. The red color of the red pigment was visible.

However, as shown below, the red dye of the separated blood cell component was visible from the transparent support side,
It is clear that there is no disadvantage in quantifying the activity values of T and ALT.

[Performance Evaluation Test 1] The change in the optical density of the red dye of the separated blood cell component detected from the transparent support side and the change in the optical density of color development due to the test component in the sample solution were measured.

The sample solutions described in Table 1 (... control serum,
... whole blood) was prepared / prepared.

20 μL of each sample solution was spotted on the tricot knitted fabric layer of the multilayer analytical element of Example 1, and the change in optical density when each analytical element was kept at 37 ° C. in a closed ink beta was measured. It was measured with 640 nm visible light. FIG. 1 shows a change in the measured optical density value. At this time, a change in optical density value from 3.5 minutes to 5 minutes after spotting was obtained, and the results are shown in Table 2.

As is clear from FIG. 1, the optical density of the red dye of the separated blood cell component detected from the transparent support side is instantaneously changed (within about 10 seconds) after spotting, and thereafter detected from the transparent support side. The optical density of the separated red blood cell component does not change (sample). The color development by AST in the sample liquid starts after the optical density of the red dye of the blood cell component detected from the transparent support side changes and ends and becomes constant (sample). Therefore, as shown in Table 2, regardless of the presence or absence of blood cell components, if the active layers of AST in the sample solution are equal, the change in optical density value over a certain period of time becomes equal.

Similar results were obtained in a similar performance evaluation test using the multilayer analysis element for quantifying ALT activity in Example 2.

[Performance Evaluation Test 2] Using the multilayer analysis elements of Example 1 and Example 2, measurements were performed to evaluate the quantification accuracy of AST and ALT activity values in a whole blood test sample, respectively.

AST by adding different amounts of AST and ALT to a 7% HSA aqueous solution,
7% HSA solutions having different ALT activity values were prepared. Separately, human whole blood was centrifuged, and a certain amount of the obtained plasma was subjected to 7% HSA.
The solution was replaced with a medium AST and ALT solution, and then mixed again with a blood cell component to prepare a whole blood sample having a hematocrit value of 40% and different AST and ALT activity values.

Each of the thus prepared whole blood samples (20 μL) was spotted on the tricot knitted fabric layers of the multilayer analytical elements of Examples 1 and 2, respectively, and each analytical element was placed at 37 ° C. in a closed ink beta.
The change in optical density from 3.5 minutes to 5 minutes after spotting when the sample was held at the temperature was determined. The results are shown in FIG. 2, FIG. 3, and Table 3.

The AST and ALT activity values in Table 3 are values obtained by the solution method for the plasma components obtained after centrifuging the used whole blood sample.

From the data in Table 3 and the graphs in FIGS. 2 and 3, after the whole blood sample was spotted, the reflected optical density value detected from the transparent support (photometric surface) side of the red dye of the separated blood cell component was constant. The amount of change per unit time of the reflection optical density of color development measured from the transparent support side after the formation of the AST activity in the multi-layer analytical element for quantifying AST activity in Example 1 was calculated as the AST activity value in a whole blood sample. In the multi-layer analysis element for quantifying ALT activity of 2, A in whole blood sample
It was found that it had a good correlation with the activity value of LT.

[Performance Evaluation Test 3] The same measurement as in Performance Evaluation Test 1 was performed using the multi-layer analysis elements of Example 1 and Example 2 to measure the respective components in the whole blood test sample.
The following measurements were performed to evaluate the effect of the hematocrit value of a whole blood sample on quantitative analysis when determining the amounts of AST and ALT activities.

AST by adding different amounts of AST and ALT to a 7% HSA aqueous solution,
Three types of 7% HSA solutions having different ALT activity values were prepared. Separately, human whole blood was centrifuged, and a certain amount of the obtained plasma was subjected to the above-mentioned method.
The whole blood samples having different AST and ALT activity values were prepared by replacing the AST and ALT solutions in% HSA with the hemocyte components again. Next, after centrifuging the whole blood sample, an appropriate amount of the obtained plasma is added or removed, whereby a whole blood sample having different AST and ALT activities and having hematocrit values of 25%, 40%, and 55% is obtained. Was prepared.

Each of the thus prepared whole blood samples (20 μL) was spotted on the tricot knitted fabric layers of the multilayer analytical elements of Examples 1 and 2, respectively, and each analytical element was placed at 37 ° C. in a closed ink beta.
The change in optical density from 3.5 minutes to 5 minutes after spotting when the sample was held at the temperature was determined. The results are shown in FIG. 2, FIG. 3, and Table 3.
The obtained optical density change was converted into AST and ALT activity values based on the relationship between the optical density values in FIGS. 2 and 3 and the AST or ALT activity values. The results are shown in Table 4. In Table 4, a hematocrit value of 0% is a result of a plasma sample containing no blood cell component.

From the data in Table 4, in the multilayer analytical elements of Examples 1 and 2, after the whole blood sample was spotted, the reflection optical density of the red dye of the separated blood cell component detected from the transparent support (photometric surface) side. The reflection optical density value of the color development measured from the transparent support after the value becomes constant is the relationship between the optical density change and the activity value obtained by the same measurement method based on the value (performance evaluation test). 3 (FIGS. 2 and 3), it became clear that the AST and ALT activity values showed almost constant values regardless of the hematocrit value in each of the AST and ALT activity values.

Considering that the activity value in erythrocytes, which is the center of the blood cell component, is about 80 times that of the AST and about 15 times that of the ALT with respect to the activity value in the plasma component, the data in Table 4 is based on the Example. It also shows that substantially no erythrocyte hemolysis has occurred in the multilayer analytical elements of Example 1 and Example 2.

[Brief description of the drawings]

FIG. 1 shows that 20 μL of each of the sample solutions shown in Table 1 was spotted on the multilayer analytical element of Example 1, each analytical element was sealed, and kept at 37 ° C. in an ink beta, and a photometric surface (transparent support) was used. 5 is a graph (calibration curve) showing a change in the reflection optical density when a reflection optical density value measured from the side is measured with visible light having a wavelength of 640 nm. Indicates the number of the sample solution. FIG. 2 is a graph (calibration curve) showing the relationship between the AST activity value in a whole blood sample and the change per minute in the reflection optical density of color development of the multilayer analytical element of Example 1. FIG. 3 is a graph (calibration curve) showing the relationship between the ALT activity value in a whole blood sample and the change per minute in the reflection optical density of color development of the multilayer analytical element of Example 2.

Claims (1)

(57) [Claims] 1. A color-forming reagent layer, on a water-impermeable, light-permeable support, which provides at least a detectable optical density change as a result of a reaction with a test component in whole blood, and a blood cell component from whole blood. And a blood cell separation layer for supplying plasma to the reagent layer by separating and removing the reagent layer, a method for quantifying a test component by colorimetry using a multilayer analysis element stacked in this order, wherein the blood cell separation layer A whole blood sample is spotted on the uppermost layer of the blood cell, and the reflected optical density value of the colored dye of the blood cell component separated from the whole blood spotted by the blood cell separation layer, which is detected from the support side, is substantially constant. After that, the amount of change in the reflection optical density per unit time in the reagent layer was measured from the support side at a wavelength in a range overlapping with the reflected light indicated by the colored dye of the blood cell component, and the obtained unit time In whole blood from the amount of change in reflected optical density A method for quantifying a test component in whole blood, wherein the activity value of the test component is determined by colorimetry.